猪圆环病毒3型SYBR greenⅠ荧光定量PCR方法建立及初步应用

Preliminary application and development of SYBR green Ⅰ fluorescent quantitative PCR method for detection of type 3 porcine circovirus

为建立一种快速、特异、敏感的SYBR greenⅠ实时定量PCR方法,检测猪圆环病毒3型(PCV3)在感染猪体内的分布情况和病毒含量,本研究根据PCV3的cap基因序列设计1对引物,对反应条件、反应体系进行优化,建立了检测PCV3 cap基因的SYBR greenⅠ实时定量PCR方法,并对动物回归试验后剖检的病料组织进行了检测。结果,该方法比传统PCR方法灵敏度高100倍,可达3.04×10^2拷贝/μL,且具有良好的特异性和重复性。在不同病料组织中均可检测到PCV3,但不同感染猪、不同组织病毒检出及含量有差异,其中肝脏、肺脏、颌下淋巴结中病毒含量显著高于心脏、心包肌、肾脏等;肝中病毒拷贝数最高,达1.16×10^6拷贝/μL,表明PCV3具有一定的组织嗜性。该方法可用于PCV3的病原学监测、流行病学调查及定量研究。

To establish a rapid,specific,and sensitive SYBR green Ⅰ real-time quantitative PCR method for the detection of the distribution and virus content of porcine circovirus type 3(PCV3) in infected pigs.A pair of was designed based on the PCV3 cap gene sequence.A pair of the reaction conditions and the reaction system were optimized.The SYBR greenⅠreal-time quantitative PCR method for the detection of PCV3 cap gene was established,and the tissue of the necropsy was examined after the animal regression test.The sensitivity of the method was 100times higher than that of the conventional PCR method,the former being up to 3.04×10^2 copies/μL and the latter being 3.04×10^4 copies/μL,with good specificity and reproducibility.PCV3could be detected in the diseased tissues,but the virus content in different tissues and pigs were different.The virus content in the liver,lung and submandibular lymph nodes was significantly higher than that in the heart,pericardium,kidney,etc.The highest copy number of virus in liver was 1.16×10^6 copies/μL, indicating that PCV3has a certain tissue tropism.This method can be used for etiological surveillance, epidemiological investigations and quantitative studies.